Fluorescent spectrum correcting method and fluorescent spectrum measuring device

ABSTRACT

A fluorescent spectrum correcting method includes comparing fluorescent spectrum obtained from micro-particles labeled with a plurality of fluorescent pigments with reference spectrum to separating the fluorescent spectrum into fluorescent spectrum for each pigment, and previously measured spectrum data is used as the reference spectrum.

CROSS-REFERENCE PARAGRAPH

The present application is a continuation application of U.S. patentapplication Ser. No. 13/287,459, filed Nov. 2, 2011 which claims thebenefit of priority from prior Japanese Patent Application JP2010-252863, filed Nov. 11, 2010, the entire content of which is herebyincorporated by reference. Each of the above-referenced applications ishereby incorporated herein by reference in its entirety.

BACKGROUND

The present disclosure relates to a fluorescent spectrum correctingmethod and a fluorescent spectrum measuring device. More particularly,the present disclosure relates to a technique for separating thefluorescent spectrum obtained from micro-particles labeled with aplurality of fluorescent pigments, for each pigment.

Generally, when micro-particles such as cells, microorganisms, andliposomes are analyzed, flow cytometry (flow cytometer) is used (e.g.,see Hiromitsu Nakauchi Edition, “Cell Engineering Additional VolumeExperiment Protocol Series Flow Cytometry Freely”, Second Edition,Shujunsha Co., Ltd., Published Aug. 31, 2006). The flow cytometry is amethod of irradiating micro-particles flowing in a flow path in one rowwith laser light (excitement light) of a specific wavelength anddetecting fluorescent light or diffused light emitted by themicro-particles to analyze the plurality of micro-particles one by one.In the flow cytometry, the light detected by an optical detector isconverted into an electrical signal to be a value and statisticalanalysis is performed to determine types, sizes, structures, and thelike of individual micro-particles.

Recently, in basic medical science and the clinical field, to advancecomprehensive analysis, there are many cases of simultaneously using anumber of molecular probes. Accordingly, biological knowledge is rapidlyaccumulated, and understanding of the phenomenon of life is advanced.For this reason, even in the flow cytometry, multi-color analysis usinga plurality of fluorescent pigments has come into wide use (e.g., seeJapanese Unexamined Patent Application Publication No. 2006-230333 andPCT Japanese Translation Patent Publication No. 2008-500558).

Meanwhile, when a plurality of fluorescent pigments are used in onemeasurement in the same manner as multi-color analysis, high-sensitivitydetectors corresponding to the number of fluorescent pigments arenecessary. The light from undesired fluorescent pigments of thedetectors is confused, and thus analytical quality control decreases. Inthe flow cytometer of the related art, since only the desired opticalinformation is taken from the desired fluorescent pigments, mathematicalcorrection, that is, fluorescent correction is performed when the lightdetected by the optical detector is converted into the electrical signalto be a value.

However, in quite a few fluorescent corrections, since the lightdetected by the undesired detector is discriminated by eyes of anobserver, human error may occur, and thus it may be incorrect. For thisreason, the observer has to understand the device and has to be trainedto use the device while having knowledge of cells, fluorescent pigments,antibodies, and the like. Therefore, observers have to have highlyspecialized knowkedge.

In the related art, a spectral deconvolution method of previouslyregistering the light emission spectrum of used fluorescent labels inadvance in a computer, separating the light emission spectrum of ameasurement target into the light emission spectrum of the fluorescentlabel using the data, and determining an existence ratio of thefluorescent labels is proposed (see Japanese Unexamined PatentApplication Publication No. 2005-181276). In spectrum absorption lightmeasurement such as an infrared spectrum method, in the related art,correction or analysis of the measured spectrum is performed on thebasis of a standard spectrum or reference spectrum (e.g., see JapaneseUnexamined Patent Application Publication Nos. 2005-195586 and2009-162667).

SUMMARY

However, in the micro-particle analyzing device provided with theplurality of high-sensitivity detectors of the related art, the strayfluorescent light detected by detectors other than the desired detectoris a big problem, as well as it being necessary to preparehigh-sensitivity detectors corresponding to the number of desiredfluorescent pigments. Particularly, in the case of the fluorescentpigments to which the spectrum is close, the fluorescent correction isnot performed due to stray fluorescent light.

For this reason, even when a plurality of high-sensitivity detectors aredisposed, there is a limit to the number of simultaneously detectablefluorescent pigments in the micro-particle analyzing device of therelated art. For example, similarly to the spectrum type flow cytometer,which does not have a plurality of high-sensitivity detectors, a nextgeneration flow cytometer usable under a condition where strayfluorescent light exists is necessary.

As described above, each of the fluorescent pigments has a specialspectrum, and the spectrum information represents the characteristics ofthe fluorescent pigment itself to be important data. However, toaccurately estimate the overlap between each spectrum and to performfluorescent correction with high precision, data of a single stainsample is necessary.

For this reason, a worker has to prepare the single stain sample foreach fluorescent pigment, and the work increases according to theincrease in the number of pigments used. Accordingly, the burden on theworker increases and work efficiency decreases. The number of operationsof fluorescent correction is in proportion to substantially the squareof the number of fluorescent pigments used, and it is troublesome to theobserver. As a practical problem, the volume of a test target objectsuch as collectible blood is finite, and thus there is a case where itis difficult to produce the single stain sample for each fluorescentpigment.

In the present disclosure, it is desirable to provide a fluorescentspectrum correcting method and a fluorescent spectrum measuring devicecapable of dissolving the overlap between each spectrum with highprecision even when the single stain sample is not prepared for eachfluorescent pigment.

According to an embodiment of the present disclosure, there is provideda fluorescent spectrum correcting method including: comparing thefluorescent spectrum obtained from micro-particles labeled with aplurality of fluorescent pigments with a reference spectrum to separatethe fluorescent spectrum into a fluorescent spectrum for each pigment,wherein previously measured spectrum data is used as the referencespectrum.

In the correction method, spectrum data in which an error from a singlestain sample is equal to or less than 8% may be used as the referencespectrum.

In the correction method, the measurement date, the potential of adetector, the type of coupled antibody, and any spectrum data of adifferent type of cell when the micro-particles are cells are used asthe reference spectrum.

In the correction method, when the micro-particles are cells,fluorescent spectrum data measured using cells may be used as thereference spectrum.

According to another embodiment of the present disclosure, there isprovided a fluorescent spectrum measuring device including: a detectionunit that simultaneously detects fluorescent light emitted frommicro-particles in an arbitrary wavelength region; an analysis unit thatseparates the data detected by the detection unit into a fluorescentspectrum for each pigment; and a memory unit that stores the fluorescentspectrum data separated by the analysis unit, wherein the analysis unituses the previously measured fluorescent spectrum data stored in thememory unit as the reference spectrum to perform separation of afluorescent spectrum.

In the device, the detection unit may be provided with a multi-channelphoto-multiplier tube.

According to the embodiments of the present disclosure, since thepreviously measured fluorescent spectrum data is used, the single stainsample is not necessary, the overlap between each spectrum can bedissolved with high precision, and further the single stain sample isnot necessary.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram illustrating a configuration of a fluorescentspectrum measuring device according to a first embodiment of the presentdisclosure.

FIG. 2A is a graph illustrating a relationship between a measurementdate and fluorescent spectrum in which a horizontal axis is a channelnumber of a detector and a vertical axis is a fluorescent intensity, andFIG. 2B is a graph illustrating an error in each wavelength (FITC:CD14).

FIG. 3A is a graph illustrating a relationship between a measurementdate and a fluorescent spectrum in which the horizontal axis is achannel number of a detector and the vertical axis is a fluorescentintensity, and FIG. 3B is a graph illustrating an error in eachwavelength (PE: CD3).

FIG. 4A is a graph illustrating a relationship between a measurementdate and a fluorescent spectrum in which the horizontal axis is achannel number of a detector and the vertical axis is a fluorescentintensity, and FIG. 4B is a graph illustrating an error in eachwavelength (spectrum corresponding to FITC of BD 7-Color Setup Beads).

FIG. 5A is a graph illustrating a relationship between a measurementdate and a fluorescent spectrum in which the horizontal axis is achannel number of a detector and the vertical axis is a fluorescentintensity, and FIG. 5B is a graph illustrating an error in eachwavelength (spectrum corresponding to PE of BD 7-Color Setup Beads).

FIG. 6A is a graph illustrating a relationship between potential of adetector and a fluorescent spectrum in which the horizontal axis is achannel number (wavelength dependent number) of a detector and thevertical axis is a fluorescent intensity, and FIG. 6B is a graphillustrating an error in each wavelength.

FIG. 7A is a graph illustrating a relationship between a coupledantibody and a fluorescent spectrum in which the horizontal axis is achannel number (wavelength dependent number) of a detector and thevertical axis is a fluorescent intensity, and FIG. 7B is a graphillustrating an error in each wavelength (FITC: CD45 vs FITC: CD45RA).

FIG. 8A is a graph illustrating a relationship between a coupledantibody and a fluorescent spectrum in which the horizontal axis is achannel number (wavelength dependent number) of a detector and thevertical axis is a fluorescent intensity, and FIG. 8B is a graphillustrating an error in each wavelength (PE: CD8 vs PE: CD3).

FIG. 9 is a density plot of blood cells for managing precision in whichthe horizontal axis is data of an antibody CD45 of a fluorescent pigmentFITC and the vertical axis is data of an antibody CD8 of a fluorescentpigment PE.

FIG. 10 is an analysis result in which the horizontal axis is data of anantibody CD45RA of a fluorescent pigment FITC and the vertical axis isdata of an antibody CD3 of a fluorescent pigment PE.

FIG. 11A is a graph illustrating a relationship between a type ofmicro-particle and a fluorescent spectrum in which the horizontal axisis a channel number (wavelength dependent number) of a detector and thevertical axis is a fluorescent intensity, and FIG. 11B is a graphillustrating an error in each wavelength.

FIG. 12A is a graph illustrating a relationship between a type ofmicro-particle and a fluorescent spectrum in which the horizontal axisis a channel number (wavelength dependent number) of a detector and thevertical axis is a fluorescent intensity, and FIG. 12B is a graphillustrating an error in each wavelength.

FIG. 13 is an analysis result in which the horizontal axis is data ofpolystyrene beads containing a fluorescent pigment FITC of BD 7-ColorSetup Beads and the vertical axis is data of polystyrene beadscontaining a fluorescent pigment PE of BD 7-Color Setup Beads.

DETAILED DESCRIPTION OF EMBODIMENTS

Hereinafter, embodiments of the present disclosure will be described indetail with reference to the accompanying drawings. The presentdisclosure is not limited to the embodiments described below. Thedescription is performed in the following order.

1. First Embodiment

Example of Method of Correcting Fluorescent Spectrum without UsingSingle Stain Sample

2. Second Embodiment

Example of Fluorescent Spectrum Measuring Device without Using SingleStain Sample

1. First Embodiment Correction Method

First, a fluorescent spectrum correcting method (hereinafter, merelyreferred to as a correction method) according to a first embodiment ofthe present disclosure will be described. In the correction method ofthe embodiment, previously measured fluorescent spectrum is used asreference spectrum when fluorescent spectrum obtained frommicro-particles labeled with a plurality of fluorescent pigments isseparated for each pigment.

Herein, the “micro-particles” widely include bionic micro-particles suchas cells, microorganisms, and liposomes, or synthetic particles such aslatex particles, gel particles, and industrial particles. The bionicmicro-particles include chromosomes constituting various cells,liposomes, mitochondria, organelles (cell organelles), and the like. Thecells include vegetable cells, animal cells, blood corpuscle cells, andthe like. The microorganisms include bacilli such as colon bacilli,viruses such as tobacco mosaic viruses, germs such as yeast, and thelike. The bionic micro-particles may include bionic polymers such ashexane, protein, and complexes thereof.

The industrial particles may be formed of, for example, organic polymermaterials, inorganic materials, or metal materials. Polystyrene, styrenedivinyl benzene, polymethyl methacrylate, and the like may be used asthe organic polymer materials. Glass, silica, magnetic materials, andthe like may be used as the inorganic materials. For example, goldcolloid, aluminum, and the like may be used as the metal materials. Theshape of the micro-particles is generally spherical, but may benon-spherical, and the size, mass, and the like are not particularlylimited.

In the correction method of the embodiment, in the spectrum data used asthe reference spectrum, an error from spectrum of a single stain sampleof measurement target micro-particles is preferably 8% or less, and morepreferably 3% or less. Accordingly, a matching error from themeasurement data is small, and it is possible to perform fluorescentcorrection with high precision.

Specifically, in the reference spectrum of each pigment, for example,the measurement date, the potential of the detector, the output oflaser, the flux of micro-particles, the type of coupled antibodies, ordata (fluorescent spectrum) of a different type of cells when themicro-particles are cells may be used. Since such conditions do not havea great influence on the fluorescent spectrum, it is possible todissolve the overlap with high precision even when such spectrum data isused in the reference spectrum to perform correction.

However, when the micro-particles are cells, the results obtained fromthe measurement using beads are not used as the reference spectrum, forexample, even when they are labeled with the same fluorescent pigment,and the opposite case is the same. As described above, even when thereis a difference in type between cells, they may be used as the referencespectrum. Of course, even when there is a difference in type betweenbeads, they may be used as the reference spectrum.

In the correction method of the embodiment, since the spectrum data inwhich the error from the single stain sample of the previously measuredmeasurement target micro-particles is 8% or less is used as thereference spectrum, it is not necessary to prepare the single stainsample at the stage of measurement. Accordingly, the burden on theworker is reduced, and thus work efficiency is also improved. Even whenthe amount of a test target object is small like a small animal such asa rat, it is possible to perform analysis without decreasing accuracy.

The fluorescent spectrum correcting method of the embodiment isapplicable irrespective of processes before and after it when the methodis a method having a process of separating the fluorescent spectrumobtained from the micro-particles labeled with the plurality offluorescent pigments for each pigment using the reference spectrum.

2. Second Embodiment Overall Configuration of Device

Next, a fluorescent spectrum measuring device according to a secondembodiment of the present disclosure will be described. FIG. 1 is ablock diagram illustrating a configuration of the fluorescent spectrummeasuring device of the embodiment. As shown in FIG. 1, the fluorescentspectrum measuring device 1 of the embodiment includes at least adetection unit 2, a memory unit 3, and an analysis unit 4, and performsthe correction method of the first embodiment. The fluorescent spectrummeasuring device 1 shown in FIG. 1 may further include a liquidtransmitting unit.

Configuration of Detection Unit 2

The detection unit 2 may have a configuration in which fluorescent lightemitted from the analysis target micro-particles can be simultaneouslydetected in an arbitrary wavelength region. Specifically, a plurality ofindependent sensors capable of detecting the wavelength region for eachwavelength region are disposed, or one or more detectors capable ofsimultaneously detecting a plurality of light such as a multi-channelphoto-multiplier tube (PMT) may be provided. The number of wavelengthregions detected by the detector 2, that is, the number of channels orsensors provided in the detector 2 is preferably equal to or more thanthe number of used pigments.

The fluorescent spectrum measuring device 1 of the embodiment may have aconfiguration in which the detector 2 is provided with a spectroscope,and the fluorescent light emitted from the micro-particles is dispersedby the spectroscope and then enters a detector such as the multi-channelPMT. The detection unit 2 may be provided with an object lens, acondensing lens, a pinhole, a band cutoff filter, a dichroic mirror, andthe like, as necessary.

Configuration of Analysis Unit 3

In the analysis unit 3, the light of each wavelength region detected bythe detection unit 2 is quantified to acquire total fluorescent lightquantity (intensity) using an electronic calculator or the like.Fluorescent spectrum correction using the reference spectrum isperformed as necessary. The result (fluorescent spectrum data) is storedin the memory unit 4.

Configuration of Memory Unit 4

The memory unit 4 stores the fluorescent spectrum data processed by theanalysis unit 3. For example, the fluorescent spectrum data of thesingle stain sample may be stored in the memory unit 4, as well as thepreviously measured fluorescent spectrum data.

Operation of Fluorescent Spectrum Measuring Device 1

Next, an operation of the fluorescent spectrum measuring device 1 of theembodiment will be described. The micro-particles analyzed by thefluorescent spectrum measuring device 1 of the embodiment are notparticularly limited, but may be, for example, cells or micro-beads. Thetype or number of fluorescent pigments modifying the micro-particles isnot particularly limited, but existing pigments such as FITC(fluorescein isothiocynate: C₂₁H₁₁NO₅S), PE (phycoerythrin), PerCP(peridinin chlorophyll protein), and PE-Cy5, and PE-Cy7 may beappropriately selected and used as necessary. The micro-particles may bemodified by the plurality of fluorescent pigments.

When the micro-particles are optically analyzed using the fluorescentspectrum measuring device 1 of the embodiment, first, excitement lightis output from a light source and the micro-particles flowing in a flowpath are irradiated with the excitement light. Then, the fluorescentlight output from the micro-particles is detected by the detection unit2. Specifically, only light (desired fluorescent light) of a specificwavelength is separated from the light output from the micro-particlesusing a dichroic mirror, a band pass filter, or the like, and the lightis detected by a detector such as a 32-channel PMT. In this case, thefluorescent light is dispersed using, for example, a spectroscope, andlight of different wavelengths is detected in each channel of thedetector. Accordingly, it is possible to obtain the spectrum informationof the detection light (fluorescent light).

Thereafter, the information of several detectors acquired in thedetection unit 2 are converted into digital signals in, for example, aconversion unit (not shown), and is further quantified in the analysisunit 3. At that time, the fluorescent correction is performed using thepreviously measured fluorescent spectrum data stored in the memory unit4 as the reference spectrum. Specifically, in the reference spectrum ofeach pigment, fluorescent spectrum data in which an error from thespectrum of the single stain sample of the micro-particles is 8% or lessis used, for example, measurement date, potential of the detector, typeof coupled antibody, or different type of cells when the micro-particlesare cells. The fluorescent spectrum data after correction is stored inthe memory unit 4.

In the fluorescent spectrum measuring device of the present disclosure,since the spectrum data in which the error from the spectrum of thesingle stain sample of the measurement target micro-particles is 8% orless is used as the reference spectrum, it is possible to perform thecorrection with high precision even when the single stain sample is notused. The fluorescent spectrum data that is the reference spectrum issequentially accumulated in the memory unit 4, and thus it is possibleto construct a database suitable for a real use situation.

Particularly, when a cell is used as a sample, there is a case where itis difficult to avoid the change of the potential of the detector andthe laser output. In such a case, in the device of the related art, itis necessary to perform the correction again to take the consistency ofthe fluorescent correction. However, in the fluorescent spectrum deviceof the embodiment, it is not necessary to do.

EXAMPLE

Hereinafter, advantages of the present disclosure will be described indetail with reference to an example of the present disclosure. In theexample, the measurement data, the potential of the detector, the typeof coupled antibody, and the type of micro-particles were changed, thefluorescent spectrum was compared, and the difference thereof wasexamined.

In the Example, an Immuno-TROL (made by Beckman Coulter, Co., Ltd.) or aMulti-Check (made by Becton Dickinson, Co., Ltd.) available on themarket as a precision managing cell was used as a sample. They arepositive process controls for flow cytometry (whole blood controlexamination target object), and represent diffused light, distributionof cell groups, fluorescent intensity, and antigen density since apositive rate of a particular surface antigen and an absolute number arecalibrated in a monocyte. A product available on the market (made bymade by Beckman Coulter, Co., Ltd. or Becton Dickinson, Co., Ltd.) wasused as an antibody labeled with a fluorescent pigment.

Dyeing of the sample was performed according to a titration method.Specifically, the temperature of the sample was kept at roomtemperature, then the antibody labeled with the desired fluorescentpigment was dropped into a dedicated plastic tube, blood of 50 μL wasdropped therein to be smoothly infiltrated, and the antibody and thecell were made to react. It was left for 20 minutes at a dark place atroom temperature. Then, a hemolytic agent (FACS Lyse solution: ammoniumchloride solution, Beckman Coulter, Co., Ltd.) of 1 ml was dropped intoit. Accordingly, red blood corpuscles were hemolyzed, granulocyte,monocyte, and lymphocyte remain. It was centrifuged and washed by anappropriate solution, and thus a high purity sample solution wasobtained.

In the measurement, the cell solution (sample solution) adjusted by themethod described above was introduced into a special measurement cellfor cell analysis formed of plastic, 3-dimensional focus was performedby a sheath solution for flow cytometer, and then it was irradiated withthe excitement light. Laser beams with wavelengths of 488 nm and 640 nmwere used as an excitement source. The fluorescent light emitted fromeach cell was dispersed by a prism spectroscope or the like, and thenwas detected by the 32 ch PMT. In the example, the 32 ch PMT was used asthe detector, but two laser beams were used as the excitement light.Accordingly, the spectrum data of 64 channels as the amount ofinformation were transmitted to the analysis unit and the memory unit.

Daily Difference

FIG. 2A, FIG. 3A, FIG. 4A, and FIG. 5A are graphs in which thehorizontal axis is a channel number (wavelength dependent number) of thedetector and the vertical axis is fluorescent intensity, and FIG. 2B,FIG. 3B, FIG. 4B, and FIG. 5B are graphs illustrating an error in eachwavelength. The florescent spectrum shown in FIG. 2A and FIG. 2B is datameasured using FITC as the florescent pigment and CD14 as the antibody,the florescent spectrum shown in FIG. 3A and FIG. 3B is data measuredusing PE as the florescent pigment and CD3 as the antibody. The same lotwas used at any date.

FIG. 4A and FIG. 4B are fluorescent spectrum of polystyrene beadscontaining the florescent pigment FITC of BD 7-Color Setup Beads. FIG.5A and FIG. 5B are fluorescent spectrum of polystyrene beads containingthe fluorescent pigment PE of BD 7-Color Setup Beads. The PMT was usedas all the detectors, and application voltage was 630 V.

As shown in FIG. 2A, FIG. 2B, FIG. 3A, FIG. 3B, FIG. 4A, FIG. 4B, FIG.5A and FIG. 5B, in spectrum A of the cell samples or beads measured byadjustment to the other date, an error from spectrum B is 8% or less,and it was confirmed that the spectrum with different measurement datewas usable as the reference spectrum. Potential of Detector

FIG. 6A is a graph illustrating a relationship between the potential ofthe detector and the florescent spectrum in which the horizontal axis isthe channel number (wavelength dependent number) of the detector and thevertical axis is the fluorescent intensity, and FIG. 6B is a graphillustrating an error in each wavelength. The fluorescent spectrum shownin FIG. 6A and FIG. 6B is data measured using the PE as the fluorescentpigment, the CD3 as the antibody, and the PMT as the detector. In theapplication voltage, PMTV150 is 525V, PMTV160 is 560V, PMTV170 is 595V,PMTV180 is 630V, PMTV190 is 665V, and PMTV200 is 700V.

As shown in FIG. 6A and FIG. 6B, the error of the spectrum was 3% orless even when the potential of the detector was changed. Accordingly,it was confirmed that the fluorescent spectrum data with the differentpotential of the detection was usable as the reference spectrum.

Type of Coupled Antibody

FIG. 7A and FIG. 8A are graphs illustrating a relationship between thecoupled antibody and the fluorescent spectrum in which the horizontalaxis is the channel number (wavelength dependent number) of the detectorand the vertical axis is the fluorescent intensity, and FIG. 7B and FIG.8B are graphs illustrating an error in each wavelength. The fluorescentspectrum shown in FIG. 7A and FIG. 7B is data measured using A: the FITCas the fluorescent pigment and the CD45 as the antibody and B: the FITCas the fluorescent pigment and the CD45RA as the antibody. Thefluorescent spectrum shown in FIG. 8A and FIG. 8B is data measured usingA: the PE as the fluorescent pigment and the CD8 as the antibody and B:the FE as the fluorescent pigment and the CD3 as the antibody. The PMTwas used as the detector, and all the application voltages were 525 V.

The data when the FITC was used as the fluorescent pigment and the CD45was used as the antibody, and the data when the PE was used as thefluorescent pigment and the CD8 was used as the antibody were analyzedusing the reference spectrum obtained from the other set of singlestain. FIG. 9 is a density plot illustrating a result thereof. As shownin FIG. 9, the analysis was performed using the reference spectrumgenerated by the single stain, and it could be divided into three cellgroups. Each group indicates that the fluorescent correction issatisfactorily performed at an orthogonal position. The number ofexistence in a region throughout a gate was FITC+PE+: 278 and PICT+PE-:750, and a ratio thereof was 0.37:1.

Then, the same analysis was performed with the data when the FITC wasused as the fluorescent pigment and the CD45RA was used as the antibody,and the data when the PE was used as the fluorescent pigment and the CD3was used as the antibody. FIG. 10 is a density plot illustrating aresult thereof. As shown in FIG. 10, in the 2-dimensionally developedplot based on the FITC and PE as the fluorescent pigment, three cellgroups are clearly classified, and each of them was positioned at theorthogonal position. Comparing distribution throughout the gate, thenumber of was FITV+PE+: 280 and PITC+PE-: 750, and the ratio thereof was0.37:1 and was equal to the existence ratio of the data shown in FIG. 9.

From the result described above, in the independent fluorescentcorrecting method using the reference spectrum, as shown in FIG. 7A,FIG. 7B, FIG. 8A and FIG. 8B, the difference of the fluorescent spectrumis 8% or less even in the different type of coupled antibody, and it wasconfirmed that the spectrum was usable as the reference spectrum.

Type of Micro-Particles

FIG. 11A and FIG. 12A are graphs illustrating a relationship between thetype of the micro-particles and the fluorescent spectrum in which thehorizontal axis is the channel number (wavelength dependent number) andthe vertical axis is the fluorescent intensity, and FIG. 11B and FIG.12B are graphs illustrating an error in each wavelength. FIG. 11A andFIG. 11B are A: fluorescent spectrum when the FITC was used as thepigment and the CD45 was used as the antibody, and B: fluorescentspectrum when polystyrene beads containing the fluorescent pigment FITCof BD 7-Color Setup Beads were used. FIG. 12A and FIG. 12B are A:fluorescent spectrum when the PE was used as the pigment and the CD8 wasused as the antibody, and B: fluorescent spectrum when polystyrene beadscontaining the fluorescent pigment PE of BD 7-Color Setup Beads wereused. All the application voltages were 630 V.

The data when the polystyrene beads containing the fluorescent pigmentFITC of the BD 7-Color Setup Beads were used, and the data when thepolystyrene beads containing the fluorescent pigment PE of the BD7-Color Setup Beads were used were analyzed using the reference spectrumobtained from the other set of single stain. FIG. 13 is a density plotillustrating the result thereof. As shown in FIG. 13, the cell groupsare classified into three, and each of them was positioned at theorthogonal position. Comparing distribution throughout the gate, thenumber of was FITV+PE+: 272 and PITC+PE-: 213, and the ratio thereof was1.28:1 and was not equal to the existence ratio of the data shown inFIG. 9 and FIG. 10.

From the result described above, in the independent fluorescentcorrecting method using the reference spectrum, as shown in FIG. 13, thedifference of the fluorescent spectrum was over 10% even when the samepigment was used between the cells and the beads, and it was confirmedthat the spectrum was usable as the reference spectrum.

As described above, according to the present disclosure, even when thesingle stain sample was not prepared for each probe, it was confirmedthat it was possible to dissolve the overlap of each spectrum with highprecision.

It should be understood by those skilled in the art that variousmodifications, combinations, sub-combinations and alterations may occurdepending on design requirements and other factors insofar as they arewithin the scope of the appended claims or the equivalents thereof.

What is claimed is:
 1. A fluorescent spectrum correcting methodcomprising: comparing a fluorescent spectrum obtained frommicro-particles labeled with a plurality of fluorescent pigments with areference spectrum to separate the fluorescent spectrum into afluorescent spectrum for each pigment, wherein previously measuredspectrum data is used as the reference spectrum.
 2. The fluorescentspectrum correcting method according to claim 1, wherein in thereference spectrum, an error from a single stain sample is equal to orless than 8%.
 3. The fluorescent spectrum correcting method according toclaim 2, wherein a measurement date, potential of a detector, a type ofcoupled antibody, and any spectrum data of different types of cells whenthe micro-particles are cells are used as the reference spectrum.
 4. Thefluorescent spectrum correcting method according to claim 3, whereinwhen the micro-particles are cells, the fluorescent spectrum datameasured using cells is used as the reference spectrum.
 5. A fluorescentspectrum measuring device comprising: a detection unit thatsimultaneously detects fluorescent light emitted from micro-particles inan arbitrary wavelength region; an analysis unit that separates the datadetected by the detection unit into a fluorescent spectrum for eachpigment; and a memory unit that stores the fluorescent spectrum dataseparated by the analysis unit, wherein the analysis unit uses thepreviously measured fluorescent spectrum data stored in the memory unitas a reference spectrum to perform separation of the fluorescentspectrum.
 6. The fluorescent spectrum measuring device according toclaim 5, wherein the detection unit is provided with a multi-channelphoto-multiplier tube.